There is a continuing need for improving biomedical implants and for improving the biocompatibility of biomedical implants upon implantation into an organism, i.e., the body. It is known that characteristics of the implant surface are strongly associated with the early events on the surface after implantation and these early events are important in determining device compatibility. It is believed that the composition, sequence, and other characteristics of proteins binding to an implant surface during the first seconds after introduction in to the organism will determine the events on a longer time scale and, ultimately, contribute to the degree of implant biocompatibility. Also, the properties of an implant surface affect the state of proteins associating with the surface, which also impacts subsequent interactions with the organism.
Typically, proteins form a monolayer on a surface in contact with the protein. See, for example, Biomaterials Science, An Introduction to Materials in Medicine, 2nd Edition, B. Ratner et al, Editors, Elsevier Academic Press (2004), p. 245, Table 1. A monolayer typically has a thickness of 2-4 nm when measured with null ellipsometry. According to de Feijter et al, “Ellipsometry as a tool to study the adsorption of synthetic and biopolymers at the air-water interface,” Biopolymers, 17:1759-1801 (1978), the monolayer thickness is converted to mass as 1 nm=0.12 μg/cm2. Accordingly, a protein monolayer on a planar surface is typically less than 0.5 μg/cm2, and often in the interval 0.2-0.5 μg/cm2. Ellipsometry is often used to study multilayers as well, but is not as accurate when used in multilayer applications as shown by Benesch et al, “The determination of thickness and surface mass density of mesothick immunoprecipitate layers by null ellipsometry and protein 125iodine labeling,” Journal of Colloid and Interface Science, 249:84-90 (2002). Also, ellipsometry can only be used on planar surfaces, and not on surfaces with a non-planar topography, which is often the case with implants. For these two reasons, protein mass per surface area is used to characterize multilayer dimension within this disclosure. Additionally, it is important to note that a device with macroscopic surface area of X may have a microscopic surface area, or nanosurface of area 10 or 100 times X, and the mass of a monolayer on a device may therefore seem much greater than a monolayer on a planar surface, due to topography and structure.
Generally, to form multilayers of a protein on a surface which are thicker than a monolayer, chemical linkers are used. For example, Tengvall et al, “Preparation of multilayer plasma protein films on silicon by EDC/NHS coupling chemistry,” Colloids and Surfaces B: Biointerfaces, 28:261-272 (2003), disclose the use of ethyl-dimethyl-aminopropylcarbodiimide/N-hydroxy succinimide (EDC/NHS) compounds to link monolayers of fibrinogen, IgG, albumin, etc., on top of one another to form multilayer films. Multilayers of different proteins having specificity for each other, such as biotin and streptavidin, or protein and antibody, have also been used to build thicker films. Fibrinogen multilayer films built by use of EDC/NHS have been used for drug delivery in conjunction with bone implants and also for maintenance of soft tissue integrity when applied to suture threads. See, for example, the Aspenberg et al US Patent Publications Nos. 2006/0002970 and 2006/0003917. Skruvcoat and T-Coat from Optovent AB are examples of commercial products employing such films, referred to as FibMat films. Chemical linkers have also been used to bind a protein film or coating to a surface, i.e., an implant surface, as well as to link together the sequential layers constituting the protein film. Chemical substances such as glutaraldehyde (GA) and silane coupling agents, for example, aminopropyltriethoxy silane (APTES), have been used for such dual purposes and for substrate binding in combination with other chemical linkers used for multilayer film formation.
The use of chemical linkers, however, to form multilayer protein films and/or to bind multilayer protein films to a substrate creates several problems. First, the process of producing such multilayer films is a time-consuming multi-step procedure. As an example, producing a fibrinogen multilayer coating using chemical linkers may take an average of 70 steps over a 2 day period, with more steps and time being required for thicker layers. Such processes clearly increase the cost of such layers in terms of both required materials and production time. Second, the use of chemical linkers introduces additional chemicals into an organism, i.e., the body, upon implantation, increasing risks of incompatibility and requiring further regulatory review and approval procedures. In fact, Sigma Aldrich, a supplier of glutaraldehyde, indicates that glutaraldehyde is toxic and environmentally dangerous, while EDC and APTES are generally considered corrosive. Further, since the chemical linkers can potentially chemically couple with a drug loaded into the film, possibly making a “new chemical entity” from a regulatory perspective, the use of multilayer films for drug delivery may be limited.
Attempts have also been made to physically manipulate proteins such as fibrinogen, lysozyme, and amyloid to form fibrils and fibers. For example, elongated proteins associate to form bands which in turn form fibrils, which are fibers of intertwined long proteins. Fibrils have been studied in order to attempt to understand protein interactions with (bio)material surfaces. See, for example, Cacciafesta et al, “Human plasma fibrinogen adsorption on ultraflat titanium oxide surfaces studied with atomic force microscopy,” Langmuir, 16:8167-8175 (2000). More specifically, fibrils of fibrinogen have been used for production of Au-nanoparticles and for stimulation of hydroxyapatite growth in the design of biomaterial surfaces. However, the processes for forming such fibrils can take extended periods of time and/or require severe reaction conditions, for example, a low pH of around 2.
Accordingly, improved multilayer protein films and/or methods of making such films are desired.